Implication of Coronin 7 in Body Weight Regulation in Humans, Mice and Flies

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1134

Functional and Molecular Characterization of Centrally Expressed Genes Associated with Obesity

ANDERS ERIKSSON

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-554-9338-7 UPPSALA urn:nbn:se:uu:diva-262479 2015 Dissertation presented at Uppsala University to be publicly examined in BMC, Husargatan 3, Uppsala, Friday, 20 November 2015 at 10:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Associate professor Christian Broberger.

Abstract Eriksson, A. 2015. Functional and Molecular Characterization of Centrally Expressed Genes Associated with Obesity. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1134. 49 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9338-7.

Obesity is a complex disorder that has reached epidemic proportions in the Western world, currently affection more than two billion people. The evidence for the genetic influence on obesity has been estimated to be as high as 70% based on twin studies. Subsequent application of genome wide association studies has identified more than 90 genes to be associated with BMI. Despite great efforts the majority of the identified genetic variants have an unknown link to BMI and lack scientific basis explaining how they affect energy balance resulting in altered body weight. This thesis aims to characterize seven BMI-associated genes, Coronin 7, Etv5, Mtch2, Nudt3, Raptor, Sh2b1 and Vps13B by performing a molecular and functional profiling in mouse, zebrafish and fruit fly. A screen analysing the regulation of the selected genes under different dietary conditions revealed altered transcript levels in mouse, zebrafish and fruit fly including a conserved regulation in all species for some of the genes. Using genetic tools the Nudt3 homolog Aps in the Drosophila CNS was knocked down and showed that Aps has a role in the regulation of insulin signaling which could explain the robust association to obesity in humans. A comprehensive in situ hybridization revealed abundant Nudt3 mRNA and protein expression throughout the brain, including in reward and feeding related regions of the hypothalamus while Nudt3 mRNA expression was significantly up-regulated in the same region of food-deprived mice. Furthermore, we were able to identify a novel molecular link between obesity and bipolar disorder. The Drosophila homologue Ets96B regulates the expression of a cellular system with links to obesity and bipolar disorder, including the expression of a conserved endoplasmic reticulum molecular chaperone complex. A connection between the obesity-linked gene ETV5 and bipolar disorder emphasizes a functional relationship between obesity and bipolar disorder at the molecular level. Furthermore, as the BMI associated genetic variants does not fully explain the heritability of obesity we decided to perform a genome wide DNA methylation profile where we compared normal-weight and obese preadolescent children. We found a CpG site located near Coronin 7 to have significantly lower methylation levels in obese children. Further studies showed Coronin 7 to be highly expressed in important brain regions involved in energy balance. Strong immunostaining was also seen in locus coeruleus, the main site for noradrenergic production, and injecting mice with an appetite suppressant increased the number of Coronin 7 neurons within the very same brain region. An evolutionary conserved metabolic function in Drosophila was also demonstrated by knocking down the Coronin 7 homologue Pod1 in the fruit fly adult nervous system.

Anders Eriksson, Department of Neuroscience, Functional Pharmacology, Box 593, Uppsala University, SE-75124 Uppsala, Sweden.

ISSN 1651-6206 ISBN 978-91-554-9338-7 urn:nbn:se:uu:diva-262479 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-262479)

We can all agree that government can't solve the obesity crisis alone. It's an ongoing issue that will re- quire a collaborative effort across private and public sectors if we want to see some long-term success.

Marcus Samuelsson

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Eriksson A., Williams MJ., Yamskova O., Cerda-Reverter JM., Schiöth HB. (2015) Nutrition regulates evolutionary conserved obesity-associated genes in mouse, zebrafish and fruit fly. Manuscript

II Williams MJ., Eriksson A., Shaik M., Voisin S., Yamskova O., Paulsson J., Fredriksson R., Schiöth HB. (2015) The obesity- linked Nudt3 Drosophila homolog Aps is associated with insulin signalling. Molecular Endocrinology 29(9): p. 1303-19

III Williams MJ., Klockars A., Eriksson A., Wiemerslage L., Voisin S., Dnyansagar R., Kasagiannis A., Ambrosi V., Fred- riksson R., Schiöth HB. (2015) The Drosophila ETV5 homologue Ets96B: Molecular link between obesity and bipolar disorder. Submitted manuscript

IV Eriksson A., Williams MJ., Voisin S., Hansson I., Krishnan A., Philippot G., Yamskova O., Herisson FM., Dnyansagar R., Moschonis G., Manios Y., Chrousos GP., Olszewski PK., Fred- riksson R., Schiöth HB. (2015) Implication of Coronin 7 in body weight regulation in humans, mice and flies. BMC Neuro- science 16: p. 13

Reprints were made with permission from the respective publishers.

Contents

Introduction ...... 11 Evolution of obesity and the obesogenic environment ...... 12 The genetics of obesity ...... 13 Central nervous system and regulation of food intake ...... 14 Obesity-associated genes ...... 17 Coronin 7 ...... 17 Etv5 ...... 18 Mtch2 ...... 18 Nudt3 ...... 19 Raptor ...... 20 Sh2b1 ...... 21 Vps13b ...... 22 Aims ...... 23 Paper I ...... 23 Paper II ...... 23 Paper III ...... 23 Paper IV ...... 24 Methodological considerations ...... 25 Ethical considerations ...... 25 Animal housing ...... 25 Feeding models ...... 25 Twenty four-hr food deprivation of mice ...... 25 High caloric diet in mice ...... 25 Ethanol injection and appetite stimulant treatment ...... 26 Housing of fruit fly ...... 26 Behavioural and metabolic studies in fruit fly ...... 26 Micronutrient diets for fruit fly ...... 26 Starvation assay ...... 26 DAMS activity assay ...... 26 CAFE assay ...... 27 Histological staining procedures in mouse ...... 27 In situ hybridization ...... 27 Immunohistochemistry ...... 28 Quantification of stainings ...... 28

Gene expression ...... 28 Tissue preparation ...... 28 Quantitative real-time PCR ...... 29 Genome wide methylation study ...... 29 Results and discussion ...... 30 Paper I ...... 30 Paper II ...... 31 Paper III ...... 32 Paper IV ...... 33 Concluding remarks ...... 36 Svensk sammanfattning ...... 38 Acknowledgement ...... 40 References ...... 42

Abbreviations

Arc Arcuate hypothalamic nucleus AgRP Agouti-related protein BAX BCL2-associated X protein BMI Body-mass index CAFE Capillary feeding CART Cocaine- and amphetamine-regulated transcript CNS Central nervous system Etv5 E26 transformation-specific variant 5 FTO Fat mass and obesity associated GFP Green fluorescent protein GWAS Genome wide association study GWM Genome wide methylation HCD High caloric diet IHC Immunohistochemistry InsP7 Diphosphoinositol pentakisphosphate InsP8 Bis-diphosphoinositol tetrakisphosphate ISH In situ hybridization LC Locus coeruleus LHA Lateral hypothalamic area MCH Melanin-concentrating hormone Mtch2 Mitochondrial carrier 2 mTORC1 mammalian protein complex known as TOR complex 1 NeuN Neuronal nuclei NPY Neuropeptide Y Nudt3 Nucleoside diphosphate linked moiety-type motif 3 OX Orexin POMC Pro-opiomelanocortin qPCR Quantitative polymerase chain reaction SH2B1 SH2B adaptor protein 1 SN Substantia nigra SNP Single nucleotide polymorphism SOLiD Sequencing by Oligonucleotide Ligation and Detection Raptor Regulatory associated protein of MTOR complex 1 VMH Ventromedial hypothalamus VTA Ventral tegmental area VPS13B Vacuolar protein sorting 13 homolog

Introduction

During the last 30 years, one of the most alarming health trends in the world has been the prevalence of obesity which has increased dramatically and shows epidemic aspects in the developed world, and becoming more prevalent in developing countries [1]. Approximately one billion adults are today considered to be overweight and an additional 300 million are obese. Obesi- ty is today one of our biggest health concerns and considered to be one of ten leading risk factors for mortality, and there are more people today dying of over-nutrition than of starvation and undernutrition [2]. As your body mass increases so does the risk for developing different diseases and numerous studies have documented the ways in which obesity and overweightness damages your health. Being overweight and obese increases the risk for developing high blood pressure, cardiovascular disease, stroke, type-II diabetes, infertility and various forms of cancer: including breast, prostate and colon cancer [3]. Moreover, the extra weight from obesity increases the load on weight-bearing joins, causing osteoarthritis. Obesity is also an economic concern. As more people become obese the financial costs in terms of health care are increasing for individuals and as well as for the society [4]. In 1998, it was estimated that obesity accounted for 6% of the total health care costs, or $42 billion yearly in the United States [4]. In 2006, a similar report con- cluded that costs had increased to 10% or close to $86 billion yearly. This number is comparable with the economic impact of cancer, which was $88.6 billion early in the US in 2011 [5]. The cut-off value for being overweight is classified as having a body mass index (BMI) equal to or above 25, and obesity is 30 kg/m2. BMI is defined as the body weight in kilograms divided by the square height in me- ters [6]. There are many more ways of measuring obesity but BMI is the most commonly used as it provides a straightforward, easy and inexpensive measure of overweight and obesity. On the contrary, it does not provide a detailed insight into the composition of the body. A person with a large quantity of muscles would get a high BMI despite having low levels of fat. Other anthropomorphic measures exist but they lack cut-off values for clas- sification for overweight and obesity. In Sweden, the prevalence for obesity is low by international comparison. Yet, 42% of adult males are overweight and 14% obese, while female obesity is slightly lower with 39% overweight and 12% obese [7]. Perhaps more alarming is that the increasing prevalence of obesity among children: 34% of

11 the children in the US are overweight and 16% obese. In Europe, specifically Sweden, the statistics are more promising. There is even a plateaued trend in the rise among childhood obesity between 1980 and 2000. Nevertheless, 25% of all boys were considered overweight and 8% obese. 22% among girls are overweight and 5% obese in 2012 [8-13].

Evolution of obesity and the obesogenic environment During the past century, our environment has dramatically changed as a result of industrialization of the western world during the 20th century. The environment has been rebuilt in terms of transportation system, land use patterns and physical design, allowing one to use less physical activity in an ordinary lifestyle. Other aspects have also changed, for example what we do during our spare time. The introduction of television and computer has altered our social behaviours with increasing amount of television affecting our health negatively. Indeed, it has been reported that children with a large amount of time in front of the TV experience an increased risk of gaining weight [14, 15]. Industrialization has also increased the abundance of cheap and high-caloric food, which often comes in oversized portions and is more aggressively marketed than healthier options [16]. A number of theories have been proposed to try to explain the genetic basis of obesity [17-19]. In an evolutionary perspective, individuals with an ability to store large amounts of energy may have been favoured by natural selection, especially during periods of famine. In today’s society this ability is of a negative char- acter due to the easy accessibility of cheap high caloric food at all times. This hypothesis is referred to as the “thrifty gene hypothesis” [19]. It suggests that obesity originated through natural selection and that some genes have evolved to protect humans from starvation. Individuals with these genes or variants were conserved during the human evolution due to the increased capability to store excess energy (body fat). The efficient use of this during times with low availability of food increased the chances for survival for the individuals that had the ability to store energy, and throughout evolution genetic variants were conserved to enhance such features. The thrifty gene hypothesis has later on been questioned since not all of us become overweight and are not equally affected by the obesogenic environment. This phenomena can be compared with other features such as large brains and upright postures [17]. A more recent theory suggest that the genetic variability have evolved without having any phenotypic impact, or until the environment became obesogenic [18]. During the early humans, obesity would have been selected against for as it would lead to a greater chance of being captured by predators. Obesity is more likely to have resulted from a genetic drift in genes that control the upper and lower limits of body fat percentage. This genetic drift putatively started when humans developed fire

12 and weapons that effectively removed predation. Predation was likely an important factor in maintaining the upper limit of the body weight. Perhaps since then, random genetic drift has resulted in a change in the distribution of body fatness due to the accumulation of predisposing genes. Supposedly, random drift, rather than directed selection, would explain why some and not most people are obese. Thus, the industrialization could have unmasked the genetic vulnerabilities for obesity.

The genetics of obesity Twin studies have determined the natural variance in BMI caused by genetic factors, and provided evidence that the genetic influence on obesity can be as high as 70% [20, 21], which means that 70% of the population variation in BMI can be attributed to genetic differences. This is a common misconcep- tion about heritability: it does not mean that, as in the case for obesity, that 70% is due to her genes and the rest is caused by the environment. High heritability means that most of the variation seen for a particular trait in the present population is caused by genetic variations. Thus, the degree of heritability (the percentage) applies to the population as a whole, and cannot be reversed and applied to individuals. The strong genetic component found for BMI is also true for other anthropometric measures of obesity, such as skin- fold thickness, waist circumference and waist-hip ratio [22-26]. Since the late 1990s, human research has dedicated much effort in identifying genetic elements associated with obesity. The early methods for identifying genes used a candidate-gene approach: exploring genetic variations in genes thought to be involved in regulation of body weight. They identified genes that followed a Mendelian inheritance pattern, but as obesity is a complex disease and likely polygenic rather than monogenic, this type of study has been less successful [27, 28]. As technology advanced, screening became possible on a genome-wide level, using millions of genetic markers. These studies are now commonly known as genome-wide association studies (GWAS). This type of study is important for the discovery of novel genetic variants associated with BMI. It typically focuses on finding associations in traits and diseases between genomic regions and search for small variations in the DNA that occur more frequently in people with a particular trait than in people without this disease. These small variations are known as single- nucleotide polymorphisms (SNPs) [29]. Most of the genetic variants identified by GWAS are intronic or intergenic. This means that the identified SNPs are more likely to affect the expression of the gene rather than the amino acid sequence (and subsequent function of the protein). A gene’s regulatory elements, possibly affected by the SNPs, do not appear to have a fixed position, but can be located before, within and also after the gene. This makes it difficult to determine which gene a SNP may be related to. In 2007,

13 the first GWAS was published and identified the fat mass and obesity associated (FTO) gene to be associated with BMI [30] and was also later confirmed [34]. So far, GWAS have identified more than 90 loci associated with BMI, and may affect the function of genes such as ETV5, MTCH2, NUDT3, RAPTOR, SH2B1 and VPS13B [31-34]. These genes will be further dis- cussed in a later section.

Central nervous system and regulation of food intake The body has an amazing capacity to maintain a stable weight despite variations in daily energy intake and expenditure [35-38]. This energy homeostasis is tightly regulated and involves complex processes with constant communication between peripheral organs, for example the liver, pancreas, and the central nervous system where hypothalamus is the main control centre (Figure 1) [39]. However, disruptions in the system controlling energy homeostasis can lead to a faulty regulation of body weight and eventually lead to obesity which is the result of a continuous imbalance between the energy intake and expenditure [35-37]. The major brain region for integrating nutritional information from the peripheral organs is the hypothalamus. The hypothalamus is anatomically divided into discrete clusters of neurons inter- connected by axonal projections that form neuronal circuits. The nutritional signals are mediated as circulating hormones and metabolites as well as neural pathways from mainly the brain stem and originate from the gustatory system and the gastrointestinal tract, as well as the pancreas, liver, muscle, and adipose tissue. All of these peripheral organs are in tight bidirectional communication with the brain and this communication is mediated either through neural connections or by hormones and metabolites. Within the hypothalamus, the arcuate nucleus (ARC) is the major projection site for the peripheral signals including leptin, insulin, ghrelin and glucose but also nutritional information [40]. ARC contains two types of neurons that express both orexigenic peptides like NPY and agouti-related protein (AgRP) and neurons expressing anorexigenic peptides like pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) [41, 42]. The later hypothalamic area (LHA) expresses two orexigenic peptides, melanin-concentrating hormone (MCH) [43] and orexin (OX) [44]. These two cell type’s acts as the main integrators of various nutritional information and circulating hormones. The anorexigenic neurons are inhibited by ghrelin and activated by leptin and insulin. Leptin and insulin carry a dual function as they both activated anorexigenic neurons and inhibit orexigenic ones. Ghrelin is also involved in the activation of orexigenic neurons. In turn, the AgRP/NPY and POMC/Cart neurons project to secondary neurons where the major projection sites are populations of neurons in the lateral hypothalamic area (LHA) and the paraventricular nucleus (PVN). Along with regulating

14 food intake the LHA and PVN also affect various autonomic responses and endocrine responses.

Figure 1. Schematic representation of the hypothalamic regulation of whole-body energy balance and metabolism. The complex and dynamic interplay between the peripheral organs and central brain regions involve a set of specific nuclei in the hypothalamus that respond to alterations in food availability as well as nutritional stores and requirements. This information is communicated by hormonal, satiety and adiposity signals from peripheral organs signalling about the nutritional state in the hypothalamus. Key peripheral organs include the pancreas, adipose tissue, muscle and liver. The hypothalamus responds accordingly to this information and induces changes in behavior regarding appetite, satiety, food intake, energy expenditure and metabolic processes. Illustration is inspired by López et al. [39].

In addition to the hypothalamus additional brain regions have been demonstrated to be involved in controlling food intake. Such regions include other non-hypothalamic brain regions, for example amygdala, prefrontal cortex and also locus coeruleus.

15 Locus coeruleus (LC) is the main site for production and release of noradrenalin throughout the central nervous system including the brain stem, cortex, thalamus, amygdala, hippocampus, hypothalamus, and spinal cord [45]. As LC innervates such a huge diversity of brain regions it has also been related to widespread functions. Some of the most important functions of LC are arousal and sleep-wake cycle, attention and memory, stress, cognitive control but also emotions, posture and balance [45]. However, of notable interest LC has been found to be involved in different processes associated with metabolism and energy regulation (Figure 2). It is known that LC and norepinephrine is affecting different aspects of feeding by methods that induce appetite, for example an intraperitoneal injection of ethanol [46, 47].

Figure 2. Schematic representation of the projections of LC in the brain. LC is the main noradrenergic centre with extensive axonal branching to diverse remote brain regions, including the cortex, hippocampus, olfactory bulb, amygdala but also thalamus and hypothalamus.

The role of locus coeruleus and noradrenaline and its effect on food intake has been studied in rat, both exogenous and endogenous noradrenaline [48, 49]. Based on pharmacological studies it has been reported that endogenous noradrenaline is involved in the control of feeding, specifically in elicitation of feeding while other studies have shown that drugs that increase endogenous noradrenaline supress eating [50, 51]. Several anti-obesity drugs are acting on noradrenaline: for example Sibutramine but also amphetamine and phentermine [52, 53].

16 Obesity-associated genes

Coronin 7 The Coronin family is an evolutionary conserved protein family, ranging from yeast to mammals, and most organisms have more than one Coronin gene [54, 55]. While Coronin proteins have a variety of functions, most of these are secondary to their actin-cytoskeleton binding ability [56-60]. Common structural features between Coronin proteins include a seven blade β-propeller formed by a WD-40 repeat-containing N-terminal domain, and also a C-terminus with a unique region to each specific Coronin, and also a C-terminal coiled coil region [61, 62]. Most eukaryotes express two different subgroups of Coronin proteins: short and long [63]. The long form of Coro- nin contains two complete copies of the basic WD-40 repeat Coronin motif but lacks the coiled-coil domain. Exactly what functional aspects the second WD40 repeat carries is currently unknown. Common for Coronin proteins is that they have an actin-binding capacity mediated through the WD40 repeat [64]. Studies have shown that Coronin proteins are involved in vesicular trafficking, cell division and also establishment of cell polarity; all of these functions are mediated through the WD40 motif [56-58]. The coiled-coil region participates in their dimerization, necessary and sufficient for its peripheral localization [65]. Distinct from other family members, Coronin 7, including its orthologues in C. elegans, and D. melanogaster, has two duplicate WD domains in tan- dem repeats [60, 66]. Furthermore, unlike other Coronin proteins, mammalian Coronin 7 is not an actin-binding protein but instead locates to the Golgi complex and is thought to be involved in Golgi complex maintenance and morphology, as well as regulation protein trafficking in an anterograde direc- tion [60, 67]. The unique feature to bind to the Golgi complex is mediated through a 47 amino acid long proline-, serine- and threonine- enriched region in the intermediate part of the protein [63]. Yet, similar to other Coro- nin-like proteins the Drosophila Coronin 7 orthologue Pod1 localizes to the tips of growing axons, where it crosslinks actin and microtubules . Overex- pression of Pod1 greatly remodels the cytoskeleton to promote dynamic neu- rite-like actin-dependent projections [63, 68]. Currently, only one study has investigated the Coronin 7 distribution in the mouse brain, where it was shown to be expressed in the hippocampus and cortex throughout development. No other brain areas were investigated in that study [60]. Moreover, an expression profile of Coronin 7, obtained using Western blot of embryonic murine tissue suggested it was ubiquitously expressed in peripheral organs [60]. Nevertheless, even after these studies it is still unclear which cell type(s) express Coronin 7 in vivo.

17 Etv5 Etv5 belongs to the Ets gene family, identified in mice during the early 1990s [69]. Common for these transcription factors is an 85 amino acid long DNA binding sequence known as the ETS domain, sharing 95% identity among the different proteins within the Ets gene family. Etv5 codes for a transcription factor important for the regulation of multiple genes involved in cell proliferation, developmental and pathogenic processes. It is an oncogene showing associations to several forms of cancer, including prostate cancer [70]. However, it was proposed as an important obesity-related locus by Thorleifsson et al, in 2009, who revealed a strong association of ETV5 with human obesity [34]. Subsequently, these results have since then been replicated in several studies using different cohorts [31, 71, 72]. In fruit fly and zebrafish, the gene is represented by the homologues Ets96B and Etv5, respectively. Almost nothing is known about Ets96B or Etv5 in zebrafish; not a single article has been published on Ets96B and only one study has been performed on Etv5 in zebrafish. That study showed an increase in the proliferation of ventral mesoderm cells as well as defects in the formation of vascular endothelium and hematopoietic cells after knocking down Etv5 [73]. In mice, Etv5 reacts to changes in nutritional status by demonstrating altered mRNA levels after ingestion of a high-calorie diet, as well as after food restriction [74-77]. Furthermore, knocking out the gene in mice reduces the body weight in postnatal males compared to control mice [74, 76]. An interesting and promising link between Etv5 and obesity has been inferred from studies in C. elegans. The orthologue for Etv5 in C.elegans, Ast-1, is vital for the development of dopaminergic neurons, as it is involved in the expression of genes that determine dopaminergic cell fate, including tyrosine hydroxylase. Ast-1 also regulates the expression of multiple genes involved in biosynthetic enzymes and transporters related to dopamine [78]. This dopaminergic link could also occur in mammals as Etv5 is highly expressed in structures in the midbrain expressing dopaminergic neurons that are involved in food related behaviours, such as the arcuate nucleus (Arc), ventromedial hypothalamus (VMH), substantia nigra (SN) and the ventral tegmental area (VTA) [76, 79].

Mtch2 Mitochondrial carrier 2 (MTCH2) was first associated to BMI in 2009 by Willer et al [31]. These results have been replicated in several studies, along with data providing evidence for MTCH2 to be associated with emotional eating in men and women [31, 34, 80-82]. In humans, evidence has been found that suggest MTCH2 to play a role in cellular processes underlying obesity. It is highly expressed in adipose tissue with elevated levels in obese women, but not in men. These altered transcript levels are not affected by

18 weight loss after bariatric surgery or behavioural modifications. Mtch2 encodes a conserved protein that belongs to the mitochondrial carrier protein (MCP) family. The general function of MCPs is to catalyse the exchange of solute across the inner mitochondrial membrane [83, 84]. Unlike other proteins in the MCP family, Mtch2 is located on the surface of the mitochondria, rather than the inner mitochondrial membrane, where it co-localizes with a large protein complex containing two proteins involved in regulating apoptosis, tBID and BCL2-associated X protein (BAX) [85, 86]. Recruit- ment of tBID to the mitochondria is dependent on MTCH2 and plays a vital role in tBID induced cell death and facilitated apoptosis [87]. Apoptosis is a key process in regulation physiological growth control and during obesity there is an increase in fat cell renewal. A highly regulated and controlled regulation of apoptosis is of fundamental importance as most both obese mice and humans experience increase in adipocyte apoptosis [88-91]. It is believed that an excess in food intake may disturb the respiratory capacity and prepare the mitochondria to induce apoptosis [92]. In agreement with this it has been shown that obese humans and rodents are experiencing an increase in apoptotic protein levels as well as cell death of adipocytes [88].

Nudt3 Nucleoside Diphosphate-Linked Moiety X Motif 3, NUDT3, belongs to the Nudix hydrolase family, widespread among eukaryotes, bacteria, archaea and viruses, and commonly classified as “housekeeping genes” involved in the regulation of important signalling nucleotides and their metabolites [93]. NUDT3 was first linked to obesity measures by the GIANT consortium, which associated the nearby SNP to BMI [32]. Interestingly, a variance in sexually dimorphic BMI has also been discovered, associating Nudt3 with an increase in the relative amount of subcutaneous fat mass in women but not men [94]. Several of the proteins within the Nudix family have been found to have significant roles in human health and disease, however, the role of NUDT3 in obesity is not yet defined [32, 94, 95]. Common for all the Nudix proteins is that they hydrolyse X-linked nucleoside diphosphates but their substrate preference varies greatly and includes nucleoside triphosphates, coenzymes, nucleotide sugars and dinucleoside polyphosphates, which suggest an involvement in diverse metabolic processes [95]. Nudix hydrolases are usually small proteins, 16-21 kDA. Enzymatic activity is mediated through a conserved 23-amino acid Nudix motif and substrate specificity is mediated through side chains and motifs elsewhere in the structure [96]. Among bacteria, there is a correlation between the number of Nudix genes and size of the genome. Streptomyces coelicolor carries about 30 Nudix genes; whereas extracellular parasites (for example Borrelia, with a consid- erably smaller genome) have none. Humans have at least 24 Nudix genes [97]. NUDT3 hydrolyses both diphosphoinositol pentakisphosphate (PP-

19 InsP5, also known as InsP7) and bis-diphosphoinositol tetrakisphosphate (PP2-InsP4, or just InsP8) [98, 99]. Moreover, Nudt3 possess mRNA decapping activity. Decapping is an important process of degradation of mRNA and involves cleavage of the 5’ cap structure and constitutes a critical step in the turnover of mRNA and is a site of numerous control inputs [100]. As Nudt3 is expressed in the cytoplasm this suggests that Nudt3 might be involved in cytoplasmic mRNA degeneration and regulation of gene expression through its decapping activity. In Drosophila, NUDT3 is represented by the orthologue Aps and, just as in mouse, it has a preference for InsP7, but otherwise little else is known.

Raptor Raptor is a 150 kDA protein and an essential component in a mammalian protein complex known as TOR complex 1 (mTORC1). It acts as an adaptor protein that recruits the different mTOR substrates [101-104]. The protein kinase target of rapamycin (TOR) is a highly conserved protein and a central regulator of cell growth and metabolism [105]. In mammals, the mTORC1 consists of five different components, mTOR, PRAS40, mLST8, Deptor and Raptor. An efficient binding of Raptor to mTOR is mediated through a conserved unique region in the N-terminal half of the protein [106]. The activity of mTOR is dependent and positively regulated by Raptor, demonstrated by knockdown experiments of Raptor using RNAi in mammalian cells [106, 107]. The processes regulated by the mTOR complex are many: protein syn- thesis, cell growth, proliferation, ribosome biogenesis autophagy, insulin signalling and nutrient transport [108]. This nutrient transport is of particular importance during pregnancy. Obese women have an increased risk of deliv- ering large babies and placental mTOR activity increases in such cases [109- 111]. In turn, upstream regulators of mTOR include different metabolic cues, nutrients, cellular energy and different growth factors including insulin [108]. Considering all of the effects mTOR and Raptor have on energy homeostasis and metabolism, it is surprising that only one study has associated RAPTOR with BMI in humans [33]. In mammals, Raptor is highly expressed in skeletal muscles and slightly less in brain, small intestine and kidney [112]. Within the cell, mTOR, but not Raptor, is expressed in the cytoplasm, lysosomes and cytoplasmic gran- ules [113-115]. However, as Raptor and the mTOR complex are highly inte- grated, it is likely that their expression is overlapping. Deleting raptor specifically in the adipose tissue of mice results in leanness and at the same time creates a better metabolic profile with enhanced glucose tolerance and resistance to diet-induced hypercholesterolemia compared to control mice [116]. Raptor knockout mice are also resistant to obesity, most likely due to a higher energy expenditure resulting from uncoupled mitochondria.

20 Sh2b1 Most of the SNPs associated with obesity are either intronic or intergenic. However, Src-homology 2B adaptor protein 1, (SH2B1) is unusual as the main SNP is actually exonic [31, 32, 34, 81, 117, 118]. Furthermore, compared to many other obesity-genes Sh2b1 had an established functional link to energy balance and obesity before it was associated to BMI by GWAS. SH2B1 belongs to an evolutionarily conserved family known as SH2B family. The mammalian SH2B family contains three members all sharing a char- acteristic pleckstrin homology domain and an SRC homology domain. Com- pared to mammals, insects only have one SH2B gene known as Lnk. In mammals, Sh2b1 is expressed in the brain in important energy-regulation regions such as the hypothalamus but also in peripheral tissues such as the heart, liver, adipose tissue and pancreas [119, 120]. The gene can be spliced in four different ways and these different splice variants are represented in different tissues [120]. SH2B1 acts as an adaptor protein regulating several different tyrosine kinases. It also functions as a modulator of the JAK-STAT signalling pathway where SH2B1 enables recruitment of target proteins (STAT) to JAK [120]. SH2B1 also appears to be able to increase the kinase activity of JAK by up to 20 times [121, 122]. The JAK-STAT is a major intracellular signalling pathway with transcriptional targets in the nucleus, including multiple hormones, leptin, insulin, cytokines and receptors for growth hormones. In the absence of leptin, SH2B1 binds to inactivated and unphosphorylated JAK at the leptin receptor. Upon this binding, target proteins are recruited which in turn activates and enhances the activity of JAK. Through the actions of leptin, the JAK-STAT pathway plays an important role in appetite regulation and energy homeostasis, resulting in several metabolic syndromes among humans caused by mutations in SH2B1, including obesity as a result from loss of function mutations of the SH2B1 gene [123]. Sh2b1 is also involved in insulin signalling through two modes of action: increasing the insulin receptor’s catalytic activity and protecting against dephosphorylation of insulin receptor substrate proteins. Deletion of SH2B1 in mice causes obesity and hyperlipidaemia, most likely due an increase in food intake resulting from impaired leptin signalling. Surprisingly, at the same time as knockout mice develop obesity, they also experience elevated energy expenditure [124, 125]. SH2B1 knockdown mice also develop age- dependent hyperinsulinemia, hyperglycaemia and glucose intolerance [126]. In fruit fly it has been found that Lnk, the Drosophila orthologue for SH2B1 shows a conserved function similar to mammals regarding its regulation of growth, glucose metabolism and energy metabolism. Disruption of Lnk promotes insulin-like signalling and increases lipid levels and energy con- servation in flies.

21 Vps13b The VPS13B gene encodes a large protein containing 3997 amino acids and 62 exons, lacking known homologies to other mammalian proteins [127]. VPS13B codes for a protein supposedly involved in protein sorting and cellular trafficking, based on strong homology to Saccharomyces cervesiae Vps13p. Vps13p regulates vesicular transport of membrane proteins between the prevacuolar compartment and the trans-Golgi network [128, 129]. In agreement with this proposed function, several studies have provided evidence that strengthens this theory. Vps13B strongly localizes to the cis-Golgi matrix protein GM130, mediated by its C terminus [130]. Using RNAi to deplete HeLa cells of Vps13B caused loss of Golgi structure, suggesting Vps13B to be involved in maintenance and morphology of the Golgi complex [130]. Additionally, VPS13B regulates the formation of membrane tubules which is in line with the proposed function in intracellular membrane traffic [130]. A brief characterization of Vps13b has been performed in the adult murine brain and identified the highest expression levels in neurons of cortical layers II-IV. Based on this expression pattern, it was also suggested that Vps13B is involved in late brain development [130]. Mutations causing loss of function in the gene lead to an autosomal reces- sive disease known as Cohen syndrome in humans [127, 131]. Common symptoms for people with Cohen syndrome include developmental delay, intellectual disability but also obesity. It has also been found that VPS13B mutations are associated with protein glycosylation defects [132].

22 Aims

Obesity is a highly complex disorder but collective data from twin and GWA studies has shown a strong genetic component and identified several genes with obesity. However, the molecular mechanism for the majority of the identified obesity genes remains unknown. The overall aim of this thesis was to provide a basic understanding for a selection of obesity-associated genes and investigate their involvement in energy homeostasis via molecular and functional characterization.

The specific aims of the studies in this thesis are:

Paper I The aim of this study was to measure the transcript levels for Etv5, Mtch2, Rptor, Sh2b1 and Vps13b in response to different nutritional status in mouse, zebrafish and fruit fly. Moreover, we wanted to provide a comprehensive expression profile of the chosen genes in mouse and fruit fly, using quantitative PCR and microarray expression levels.

Paper II This study focused on describing the expression pattern of Nudt3 as well as its cellular localization and abundance in the CNS and peripheral organs. Furthermore, we wanted to determine if it had any effect on metabolism, so we performed a phenotypical characterization of fruit flies lacking the Dro- sophila Nudt3 orthologue Aps.

Paper III In Paper III we provide an expression profile of ETV5 in mouse brain and periphery by means of in situ hybridization and immunohistochemistry. The expression pattern of the Drosophila orthologue Ets96B is also characterized using genetically driven green fluorescent protein (GFP) staining. We also wanted determine possible gene networks using SOLiD sequencing of the entire transcriptome in Ets96B knockdown flies. Furthermore, we knocked down Ets96B to investigate basic behaviour pattern with respect to metabolism and general activity.

23 Paper IV In Paper IV we sought to further explore the functional genomics of obesity by performing a genome wide methylation profile in obese and normal weight pre-adolescent children. We also characterized the expression pattern of the differently methylated gene Coronin 7 in mouse brain and peripheral organs.

The projects included a variety of experiments and techniques, which include gene expression analysis using qPCR, immunohistochemical detection and in situ hybridization of the genes of interest, and characterization of knockdown models in fruit fly. The studies were performed in well- established and relevant model organisms: mouse, zebrafish and fruit fly.

24 Methodological considerations

Full description and more detailed experimental procedures used in this thesis are provided in each of the individual papers. The following sections briefly describe the methods used the most.

Ethical considerations All animal procedures were approved by the Uppsala Animal Ethical Com- mittee). All experiments followed the guidelines of Swedish legislation (An- imal Welfare Act SFS1998:56 and European Union legislation, convention ETS123 and Directive 86/609EEC).

Animal housing Mice were housed in groups of maximum 8 unless other stated. They were housed in standard Macrolon type III cages at constant temperature (23 ± 1 °C), humidity (50 ± 5%) and a 12:12 dark-light cycle.

Feeding models Twenty four-hr food deprivation of mice Male, C57BL/6J mice had unlimited access of chow to just before the onset of darkness when the food was removed. 24 hours later the animals were sacrificed. Relevant tissues were collected and immersed in RNAlater within 10 minutes after sacrifice to avoid breakdown of RNA. Control mice had ad libitum access to chow. Both food deprived mice and control mice had access to water at all times.

High caloric diet in mice To induce obesity in male, C57BL/6J mice were placed on an atherogenic high-caloric diet (R638, Lantmännen, 0,15% cholesterol, 21% fat). The mice were sacrificed after eight weeks and at this point they had received a highly significant increase in weight compared to control mice. Relevant tissues

25 were collected and immersed in RNAlater within 10 minutes after sacrifice to avoid breakdown of RNA. Control mice had ad libitum access to standard chow and water.

Ethanol injection and appetite stimulant treatment Mice were injected with saline or 2.0 g/kg b. wt [15% (w/v)] ethanol by intraperitoneal injection. Perfusion of mice was performed 1.5 h later.

Housing of fruit fly All flies, unless otherwise stated, were maintained on Jazz mix standard fly food (Fisher Scientific), supplemented with 5% Brewer’s yeast extract (VWR). Flies were maintained at 25ºC in an incubator at 60% humidity on a 12:12 light:dark cycle. All assays were performed at 25 ºC, unless otherwise stated. In all assays, the GAL4 drivers and UAS transgenic flies were crossed to w1118 flies and their F1 progeny used as controls.

Behavioural and metabolic studies in fruit fly Micronutrient diets for fruit fly All diets consisted of varying concentrations (g/dl) of sucrose (Sigma, Swe- den) or yeast extract (VWR, Sweden) in 1% agarose. Newly eclosed adult males were maintained on these diets for 5 day.

Starvation assay Starvation resistance was measured by placing 20 male flies, which were 5 to 7 days old, in a vial containing 5 ml of 1% agarose, which provides water and humidity but no food source. The vials were kept at 25°C in an incubator, on a 12 h:12 h light:dark cycle. The numbers of dead flies was counted every 12 hours. This allowed for the calculation of the median time of death and survival rate. At least 10 replicates for each genotype were conducted.

DAMS activity assay Flies were tested using the Drosophila Activity Monitorign System (DAM2, TriKinetics). Briefly, flies were placed in 5 mm x 65 mm glass tubes during the morning, with food placed in one end of the tube and sealed with a piece of cotton in the other end. Flies were continually monitored by the use of a

26 light beam, with the number of breaks continuously recorded over a 48 h period.

CAFE assay This method was modified from Ja et al. 2007 [133]. A vial, 9 cm by 2 cm (height X diameter), containing 1% agarose (5cm high) to provide moisture and humidity for the flies, was used for this assay. A calibrated capillary glass tube (5 µl, VWR International) was filled with liquid food which contains 5% sucrose, 5% yeast extract and 0.5% food-colouring dye. A layer of mineral oil was used to prevent the liquid food from evaporating. Five males, which were 5-7 days old, were put inside the chamber and the open- ing of the vial was covered with paraffin tape, a capillary tube was inserted from the top through paraffin tape. The experimental set up was kept at 25oC, 50% humidity on a 12:12 hour light:dark cycle. At least 10 replicates were performed for each genotype.

Histological staining procedures in mouse In situ hybridization In situ hybridization (ISH) is a histological colouring technique used to detect and label different nucleic acids, for example mRNA, in morphological- ly preserved cells in fixed tissue. ISH was first described by Gall and Pardue in 1969 and encompasses a digoxigenin-labelled RNA anti-sense probe, complementary to the target mRNA of interest expressed inside cells. The probe is thereafter visualized in situ in the cell body. A variety of visualiza- tion methods exist, we utilized a non-radioactive enzymatic reaction with enzyme-coupled antibodies. The cells expressing the mRNA of interest are visualized by adding a substrate that becomes coloured by the enzyme- coupled antibodies. This semi quantitative technique was used in paper I-III to identify the mRNA expression as a first step to elucidate the function of these genes in the central nervous system of mice. RNA probe were produced by in vivo transcription using a RNA polymerase in the presence of a digoxigenin-substituted nucleotide, dig-UTP. The cDNA clones used for the transcription had been incorporated into known vectors and are commercial- ly available (Source BioScience, United Kingdom). In papers II-IV a single RNA probe ISH was used on coronal sections for localization for genes within the mouse CNS. The RNA probe was visualized using FastRed (Roche, Sweden) which creates a fluorescent staining.

27 Immunohistochemistry Immunohistochemistry is a techniques used for histological stainings. This technique is a powerful tool to determine the localization and abundance of proteins in a tissue sample. Immunohistochemistry utilizes the specific binding of antibody to its epitope. An un-conjugated primary antibody binds to a specific characterization located on the epitope of an expressed protein. By adding a secondary antibody which has been conjugated with a fluoro- chrome one can localize, visualize and amplify the localized protein. This technique enables the use of two and even three different primary antibodies simultaneously on the same cell or tissue section. A double IHC allows for comparative studies with proteins with unknown localization and proteins with known localization and cellular function. In this thesis, immunohistochemistry was used to determine the abundance and localization of the novel genes of interest in mouse brain. For each of the antibodies used a comprehensive optimization of the IHC proto- col to avoid background and unspecific staining. This optimization allowed improvement of the signal by adjusting the blocking reagents, type of secondary antibodies and the concentration of the previously mentioned components to reduce the background and maximize the intensity of the staining. The advantage of being able to combine two different antibodies and detect both genes and proteins at the same time was used in paper II-IV.

Quantification of stainings All quantifications in this current thesis have been performed manually after ISH or immunofluorescence. For the immunofluorescent stainings used to analyse the abundance of immunostaining in a number of major brain areas a certain amount of cells were counted. At least three different animals were used in these experiments. For the ISH, only cells with a complete staining around the nucleus were counted to give a confident approximation of the intensity of the staining. This intensity was then graded on a scale from “–“meaning no staining to “+++” which represents intense staining.

Gene expression Tissue preparation Samples to be used for quantitative real-time PCR was dissected on ice and immersed in RNAlater (Fisher Scientific) before freezing at -80°C until further analysis.

28 Quantitative real-time PCR Compared to normal polymerase chain reaction (PCR), quantitative real-time PCR is a measurable method with the advantage of detection the amount of DNA being produced after each cycle. During PCR one is able to amplify a small specific DNA sequence from a relative small amount of starting mate- rial. Quantitative PCR monitors the amplified product in real time using a fluorescent dye being incorporated into the reaction, which is proportional to the amount of product being produced. The quantitative measurement is based on calculating the number of amplification cycles need to produce a certain amount of DNA. Primers for each of the housekeeping genes and genes of interests were designed to amplify only small segments of the gene, using Beacon Designer (Premier Biosoft, USA). A specific annealing temperature was optimized for each primer prior to the experiment using temperature gradients and through analyzing PCR product melting curves, to assure as high efficiency as possible. In this thesis we used qPCR for genotyping and verification of reduced mRNA levels of knockdown flies (paper II, III and IV). Additionally, we used qPCR method to determine gene expression levels for our genes of interests in relevant tissue samples collected from mice, fruit fly and zebrafish under different nutritional statuses (paper I-IV). RNA was extracted from the tissue and cDNA was synthesized by using reverse transcriptase. PCR reactions were performed in triplets and were run on iCycler temperature cyclers. Data were analysed using iQ5 software (Bi- oRad, Sweden).

Genome wide methylation study Methylation of CpG sites in the DNA constitutes an important and critical epigenetic regulation in many eukaryotes and it is one of the most studied epigenetic mechanisms. When located at a gene promoter it is often involved in transcriptional regulation where it silences the gene. For the purpose in Paper IV, genomic DNA was isolated from whole blood obtained from obese and normal-weight participants. Two different linear models were used: one adjusted for age, sex, Tanner stage and white blood cell count and one where we selected the top 15 sites whose methylation levels correlated with the obesity-linked gene STK33 polymorphism.

29 Results and discussion

Paper I To facilitate the understanding on how genetic variation affect the development of obesity and contribute to increased body weight we analysed the regulation in some obesity-associated genes in response to different dietary conditions. For the purpose of this study, we decided to analyse expressional changes for the following genes: Rptor, Etv5, Mtch2, Sh2b1 and Vps13b. In addition to being associated with obesity they also carry functional homologues in mouse, zebrafish and fruit fly. We also performed a comprehensive expression pattern analysis in mouse and fruit fly. Quantitative PCR showed ubiquitous expression pattern in peripheral organs and also in the brain for all of the genes included in this study. Using microarray expression data downloaded from FlyAtlas we created an expression profile for flies. The expression pattern in fly was in agreement and confirmed the wide expression pattern seen in mouse. As most of the genetic variants found in GWAS are typically intronic or intergenic means that they more likely to affect the transcriptional regulation rather than the amino acid sequence and thereby the function of the protein. It has also been shown that energy regulating peptides expressed in the hypothalamus show different mRNA expression levels during different dietary paradigms. To determine the effects of these genes on relevant brain structures and key peripheral organs important in metabolism we performed a screen of the associated genes in mouse, zebrafish and fruit fly and analysed the levels of these genes during different nutritional states. All but Sh2b1 were found to be affected by nutritional status. Many were regulated by either fasting or by consumption of the high caloric diet. More specifically, we found upregulation of Etv5 transcript levels in the hypothalamus and liver from food deprived mice. In fruit flies, increased transcript levels of Ets96B were observed after 9 and 24 hours of starvation in the central nervous system. In liver tissue, Etv5 was upregulated in mice after 24h of starvation as well as in zebrafish after one and two week’s starvation. This conserved regulation further strengthens Etv5 to be important for the etiology of obesity. As Etv5 is a transcription factor it is likely that it might influence the energy balance by transcriptional regulation of certain genes linked to food intake. Furthermore, Rptor, Mtch2 and Vps13b were all upregulated in the liver of obese mice, while food deprived zebrafish had increased levels of

30 Vps13 in the liver from starved animals. An opposite response was seen in food deprived flies which showed reduced transcripts levels of Vps13 and Raptor.

Paper II In paper II we take our starting point in profiling the expression pattern of Nudt3 mRNA in mouse peripheral tissues and seven coronal cross sections encompassing the entire brain using quantitative real-time PCR analysis. Nudt3 was found to be expressed in all tissue samples with the highest expression levels in the testis and the lowest in the blood cells. The cerebellum, brain stem and intestines also had high levels of Nudt3 expression. ISH eval- uated Nudt3 mRNA distribution in the adult male mouse brain. Ubiquitous and widespread expression of Nudt3 was observed in the entire brain, with expression in both white and grey matter. Our ISH also showed Nudt3 to be highly expressed in the amygdala, hypothalamus and striatum, regions that have important roles in regulating food intake and energy homeostasis. To identify cell-types expressing this protein and the abundance of Nudt3 protein in different brain regions we used immunohistochemistry. Five major brain areas were co-stained with an antibody for Nudt3 together with different markers for two subsets of neurons: inhibitory and excitatory ones. We showed that Nudt3 immunoreactivity is mainly in neuronal cells and that Nudt3 is expressed in more excitatory neurons than inhibitory neurons. In- terestingly, hypothalamus had the highest number of excitatory neurons that expressed Nudt3. Considering that Nudt3 is presented as a gene associated with obesity measures it is interesting that we find Nudt3 positive neurons to such high degree in this region. In this study we also analysed the transcript levels in mice that had been subjected to different feeding paradigms: 24 hour food-deprivation, obese and normal control mice. Depriving adult male mice of food for 24-hours resulted in an increase in Nudt3 transcript in the hypothalamus, the brain stem and the liver. We next sought to explore the potential role of Nudt3 as a regulator for metabolic homeostasis in Drosophila by knocking down the Drosophila homolog, Aps, in the central nervous system in fruit flies. We employed a starvation assay: a technique uncovers any disruption in feeding behaviour, lipogenesis, lipolysis or gluconeogenesis in flies. Flies deficient of neuronal Aps displayed a susceptibility to starvation while inducing hyperphagia. Concurrently, neither starvation nor the macronutrient content affected the transcript level of Aps. The lipid levels in knockdown males had a trend of being elevated compared to its control. However, during the starvation the knockdown males displayed increased lipid levels. These results indicate that loss of Aps in neurons inhibits the recruitment of lipids during starvation. Next, we sought to measure the glucose and trehalose levels in adult

31 males. Knockdown flies had significantly decreased circulating glucose and trehalose levels both before but also after 12h and 24h of starvation. Interest- ingly, the body trehalose levels were higher before starvation, but during starvation no difference was observed. Glycogen levels displayed no differences before starvation but were significantly lower at 24h starvation. Aps knockdown flies have significantly lower circulation carbohydrate levels during ad libitum, together with higher levels of stored carbohydrates indicate that neuronal Aps regulates feeding, lipid and sugar homeostasis. We were also able to find that neuronal Aps regulates insulin signalling. Aps knockdown flies have a decreased expression of Ilp3, Ilp6, and Akh, three genes involved in the insulin-glucagon-system. Furthermore, knocking down Aps in insulin producing cells in the brain reduces the carbohydrate levels under starvation conditions. It also decreases glycogen levels after 12h and 24h of starvation and increased the body trehalose levels before starvation but decreases them during starvation.

Paper III Bipolar disorder (BD) is associated with obesity, overweight and abdominal obesity. Several physiological and behavioural similarities exist between obesity and bipolar disorder. For example, obese people are more impulsive and experience heightened reward responsiveness, phenotypes that also can be found in people with bipolar disorder. Patients with BD have an increased prevalence of obesity, and obesity is also associated with greater severity of the bipolar disorder [134, 135]. The molecular mechanism explaining such an association is unknown. This is also true for other physiological disor- ders; obese people have been found to have several times higher rates of depression. Using Drosophila as a model organism, we demonstrate that Ets96B regulates genes involved in cellular systems associated with both obesity and bipolar disorder. We performed a SOLiD sequence analysis of the entire Ets96B knockdown fly’s transcriptome and revealed that Ets96B regulates expression of two major groups of genes involved in the mitochondrial electron transport chain and also endoplasmic reticulum localized molecular chaperones. Several studies have been able to link the electron transport chain in the mitochondria to obesity and insulin resistance [136-138]. Fur- ther, inhibiting the expression of Ets96B in the nervous system during development increases lipid storage, anxiety and impulsivity, typical phenotypes for obesity and bipolar disorder. We were also able to see increased startle response in flies when Ets96B was knocked down during development, a phenotype that disappeared when it was knocked down in adult flies. The startle-response test is similar to prepulse inhibition (PPI) test in mammals, a test that take advantage of the neurologic phenomena in which a weaker

32 prestimulus inhibits the following stronger startle stimulus. Humans with disrupted response to PPI are linked with bipolar disorder [139]. As we saw that knockdown of Ets96B affect genes involved in energy homeostasis and protein production, we wanted to see what effects loss of Ets96B have on metabolism. The transcript levels of Ets96B in the brain were increased during a 24 hour starvation period but not by the macronutrient content. When Ets96B was knocked down during development they became more susceptible to starvation. Interestingly, when knocked down in adult flies they show an opposite phenotype, with an increased resistance to starvation. However, not any of the knockdown flies showed any alterations in food intake. Next, we continued with measuring the stored lipid content before and during starvation. The similarity between the mechanisms of lipid storage control is surprisingly highly conserved from fly to man. Many of the biochemical and biological pathways are very similar. In flies the body fat is composed of neutral lipids, mainly triacylglyceride, TAG. TAG is stored in intracellular organelles of adipose tissue, known as lipid droplets. Measure the content of fat in flies can be used as an indicator of imbalances in lipometabolism homeostasis. To quantify this, we analysed the levels of TAG before and during starvation. The male flies with Ets96B knocked down during development had a significantly higher lipid content compared to controls but after 12h and 24h of starvation all strains showed lower total TAG levels with no differences between strains. These results indicate that knockdown flies display a phenotype that mimics obesity with increased lipid storage. Adult Ets96B knockdown flies had lower TAG content than control, contrary to what was seen in flies with Ets96B knocked down throughout development. We also characterized the expression profile using ISH on coronal mouse brain sections. Etv5 mRNA was expressed in a specific and distinguishable pattern in certain brain regions, predominantly the cerebral cortex, the amygdala, and the hypothalamus. In the mesencephalon expression was re- stricted to the ventral tegmental area. To analyse the expression in Drosophi- la and to see if Ets96B could be involved in the development of the nervous system, we used GFP staining by crossing Est96B-GAL4 to UAS-GFP and mapped GFP expression in the brain of third instar larvae. Analysing the expression showed the GFP staining to be located to dopaminergic neurons.

Paper IV Although GWAS have identified more than 90 genes associated with obesity, the identified loci only explain a small part of the heritability of obesity, which means that there are either many more genes to be identified or other forms of variations must be considered, such as epigenetics. The amount of evidence showing the involvement of epigenetics in complex diseases such

33 as obesity is constantly increasing. To further explore the h functional genomics of obesity and establish if there is a correlation between weight cate- gory and methylation levels of CpG sites/islands we investigated the genome-wide methylation profile using whole blood obtained from obese and normal-weight participants. Two different linear models were used to stratify the methylation data and look for a common gene designated by both linear models and only one CpG site appeared in top 15 of both linear models. We found that obese children had significantly lower methylation levels at a CpG site located near Coronin 7 encoding a tryptophan-aspartic acid dipep- tide (WD)-repeat containing protein, most likely involved in Golgi complex morphology and function. The methylation level and gene expression relationship is complex, but typically a high level of gene expression is often associated with lower methylation. Next, we characterized the expression of Coronin 7 in the mouse brain and in peripheral organs. Using qPCR we were able to show that Coronin 7 is widely distributed in both brain and in peripheral organs. Intriguingly, Coronin 7 is expressed in important food-regulation areas within the brain such as the hypothalamus, ventral striatum and amygdala. As a comparison, microarray expression data for the Drosophila melanogaster Coronin 7 homologue pod1 was downloaded from FlyAtlas. Similar to mouse, pod1 expression was ubiquitous, but had higher expression levels in the brain. We performed a detailed characterization of the Coronin 7 expression pattern in adult mouse brain using ISH and immunohistochemistry on a large number of coronal brain sections. These correlated well with results obtained from the qPCR expression profile and showed that Coronin 7 was highly expressed throughout the CNS including food intake and energy homeostasis regulation areas, such as the arcuate nucleus, the anterior hypothalamic area, posterior part and ventromedial hypothalamic nucleus. Of note, depriving adult male mice of food for 24-hours resulted in a statistically significant decrease in Coronin 7 hypothalamic expression. An immunohistochemical staining demonstrated Coronin 7 to be expressed in all major brain areas. It also revealed Coronin 7 to be highly expressed in the locus coeruleus (LC). Neurons in the LC are known to respond to the appetite stimulant ethanol. Interestingly, treating mice with ethanol resulted in an increase in the number of Coronin 7 positive neurons within the LC, compared to mice injected with saline. As the LC is the major noradrenaline synthesizing site, and many of its noradrenergic neurons project to the hypothalamus, the fact that Coronin 7 is expressed therein suggests a link between Coronin 7 and the noradrenergic regulation of the hypothalamus. To better understand if Coronin 7 could be involved in regulating metabolic homeostasis we also used the genetically-tractable Drosophila system by knocking down the homologue, pod1. We found that pod1 mutant flies displayed significantly more resistance to starvation than control flies. Flies

34 being subjected to a diet rich in both protein and sugar had significantly increased levels of pod1 mRNA expression, compared to normal fed flies.

35 Concluding remarks

Through GWAS, numerous genetic variants have been associated with increased body weight. For a few of the associated genes a clear functional role in body weight regulation has been found, but for the majority the link to energy balance remains unknown. In this thesis we demonstrate functional links for seven of these obesity-associated genes. We also show that the fruit fly can be useful for identifying irregularities in metabolism by knockdown of the gene of interest. Further, we demonstrate that results from the fruit fly can be applicable to both mice and humans. Ever since FTO was first identified in a GWAS as a BMI-associated loci the research has come a long way. At present, over 90 SNPs associated with BMI have been found, which illus- trates the complexity of the disease. The individual contribution of each allele only explains a small proportion of the variance in adult BMI, typically between 0,05 and 0,24 BMI units [31], but this is to some extent irrelevant as obesity is only in a few cases a monogenic disease. It has been shown that carrying a larger number of risk alleles increase the body weight more compared to individuals carrying few risk alleles [29]. However, the combined effect of the individual genes on BMI still does not add up to the speculated heritability of 70%. Moreover, the results from paper IV show that other forms of genetic variations should be considered, such as epigenetics (DNA methylation). It is evident that some individuals respond differently to an obesogenic environment and in some cases these responses have detrimental effects resulting in obesity which might be explained using epigenetics. Therefore, both the GWA and the genome wide methylation studies needs to be improved since they currently fail to detect all of the genetic variants. However, identifying these obesity genes is just one part of the problem, to understand the biochemical pathways that these genes are involved in are equally important. Also, the molecular mechanisms by which the BMI- associated SNPs influence their respective genes remain elusive. Each one of associated genes needs to be further analysed to unravel how they are associated with BMI and how they might contribute to the development of obesity. As multiple processes could contribute to the risk of obesity, it is important to study the properties of these genes. Further investigations are needed with regards to their expression profile, protein abundance, the effect of nutritional status on transcript levels, as well as behavioural and metabolic characterizations. The transgenic fruit fly models can be helpful for this. Hopefully this will uncover the neurological circuits and whole-body energy

36 expenditure, including the peripheral pathways involved in the regulation of energy balance. Interestingly, the strongest associations between the identified SNPs to body weight reside in intronic or intergenic regions, suggesting that they could also have a role a role in the regulation of the nearby genes. As shown in Paper I we demonstrate altered transcriptional levels in response to different nutritional states. Even though we see small and modest changes on the individual mRNA levels, it still suggests that the combined effects of each one of these small alterations could potentially lead to a sub- stantial increase in body weight. All of this will provide valuable information into how the obesity-genes relate to energy homeostasis. The need for pharmacological treatments for obesity is high as the lifestyle modifying programs developed to treat obesity have notoriously been found to be lacking desired effect. Most of them have proven to be difficult to implement and maintain on a large scale for a prolonged time. This is made even more complicated as some public health messages are perceived as stigmatizing and is actually lessening the motivation to improve one’s health [140]. With new improvements in automated phenotypical characterizations it is possible to perform large-scale high throughput drug screens in in vitro models, such as fruit fly. Using effective tools and appropriate animal models might lead to the development of new and effective pharmacological treatments for obesity. The genetic component of obesity has been extensively studied and several genetic variants been found to be associated with BMI. Despite great efforts to provide a scientific basis for how these genes correlate with obesity, the etiology of obesity is slowly being elucidated by examining genes with putative mechanisms in its development. This thesis presents results for seven of BMI-related genes and through the use of molecular techniques and model organisms; we show expression profiles and phenotypic data in mouse and fruit fly association the selected genes to metabolic irregularities.

37 Svensk sammanfattning

Under de senaste 30 åren så har prevalensen för övervikt och fetma mer än fördubblats. Fetma och övervikt är idag en av vårt samhälles största hälso- risker och räknas dessutom som en av de tio största riskfaktorerna för död- lighet. Genom att jämföra genetiska analyser över hela människans DNA i s.k. genome-wide association-studier så har forskare lyckats hitta mer än 90 gener som är associerade till BMI. Endast ett fåtal av dessa gener har en bevisad biologisk effekt som kan förklarar varför just den är kopplade till ökad kroppsvikt och BMI, men för merparten så är kopplingen fortfarande okänd. De studier inkluderade i denna avhandling fokuserar på ett urval av de gener som har en okänd koppling till ökad kroppsvikt och uppgiften har varit att studera dessa gener i det centrala nervsystem och hur de kan tänkas påverka hjärnan på ett sådant som leder till förändrad energireglering. I den första studien så fokuserade vi på sju stycken gener som tidigare blivit kopplade till fetma hos människor. Dessa gener finns även represente- rade i ett flertal andra organismer, exempelvis mus, zebrafisk och bananfluga. Trots de uppenbara skillnaderna mellan människa, mus, zebrafisk och bananfluga så finns det stora likheter; merparten av de sjukdomsorsakande generna i människa finns även i bananfluga och zebrafisk. Dessutom så an- vänder mus, zebrafisk och bananfluga likartade metaboliska vägar och har organ med snarlik funktion som hos människor. Vi undersökte hur uttrycket av dessa gener förändrades vid svält och övergödning. Vi fann att genen Etv5 uppreglerades i hypotalamus och i levern hos möss och i levern hos zebrafisk som hade fått svälta ett antal timmar respektive en och två veckor. I bananfluga så fann vi liknande resultat: efter 9 och 24 timmars svält så var samma gen uppreglerad i det centrala nervsystemet. Denna konserverade reglering av Etv5 tyder på att genen har liknande funktion i alla tre organismer och stärker dess association till BMi hos människor. Även de andra generna visade förändrade uttrycksnivåer: Rptor, Mtch2 och Vps13B var alla uppreglerade i levern hos överviktiga möss, medan zebrafisk visade ökade nivåer i levern på svultna djur. En motsatt regleringsmekanisk fann vi hos bananfluga där Vps13 och Raptor hade reducerade uttrycksnivåer i svultna flugor. I de tre följande studierna så gjorde vi mer noggranna studier på tre olika fetmagener, Nudt3, Etv5 och Coronin 7. Vi använde oss av möss för att ka- raktärisera uttrycksmönstret för dessa gener och för att undersöka om dessa gener reglerar metabolisk homeostatisk kontroll så använde vi oss av gene-

38 tiska metoder för att stänga av uttrycket av dessa gener i bananflugor (knockdown). Hos möss så visade Nudt3 ett omfattande uttrycksmönster i olika perifera organ och i hjärnan. Vi fann uttryck i regioner som är kända för att vara involverade i energireglering inklusive hypothalamus. Med immunohistoke- miska metoder så fann vi att Nudt3 är mer uttryckt i stimulerande nervceller än i inhiberande. Vi fann också att vid förändrade uttrycksnivåer i hypotalamus, hjärnstammen och lever möss utsatta för svält. Vidare så använde vi olika genetiska tekniker för att inaktiverade motsvarande gen för Nudt3 i bananfluga, Aps. Med Aps avstängd så fann vi att flugorna visade en ökad känslighet för svält samtidigt som Aps-knockdown flugor åt mer än sina kontroller. Olika metaboliska faktorer visade sig också vara påverkade. Vi mätte nivåerna för kolhydrater och fann att Aps-knockdown flugor hade lägre nivåer av kolhydrater under svält. Utöver detta så har också Aps- knockdown flugor en minskad förmåga att rekrytera lipider under svält. Dessa flugor var också mer känsliga för svält än normala flugor. Samtidigt som knockdown flugor var mer känsliga för svält så fann vi även att de åt mer än kontrollflugorna. Alltsammans så tyder detta på att Aps i bannafluga är involverad i regleringen av insulinsignaleringen. I den tredje studien så fann vi att Etv5/Ets96B reglerar cellulära system som är kopplade till både fetma och bipolär sjukdom. Inhibition av uttrycket av Ets96B i bananfluga ökar triacylnivåerna, startle-reflexen sam orsakar hyperaktivitet och impulsivitet, fenotyper som är associerade både till bipo- lär sjukdom och fetma. Vi visar också att Etv5 är uttryckt i hjärnregioner som är involverade i både dopaminfrisläpp men också födointagsreglering. Samma sak finner vi i bananfluga, där vi visar att Ets96B är uttryckt i dopa- minproducerande celler. De gener man hittills hittat kan inte helt förklara den totala ärftligheten för fetma vilket innebär att det måste finnas andra typer av mekanismer som styr ärftlighet, exempel på sådana mekanismer inkluderar epigenetiska föränd- ringar. Epigenetik innebär fenotypiska förändringar som sker oberoende av förändringar på DNA-strängen. En av de vanligaste epigenetiska mekan- ismerna är metylering av DNA-basen cytosin. Vi genomförde en profilering av metyleringsmönstret hos normalviktiga och överviktiga ungdomar och fann att genen Coronin 7 hade lägre metyleringsnivåer hos överviktiga barn. Normalt sett så tyder en lägre metylering högre uttryck av genen. Vidare så visade vi att Coronin 7 är uttryckt i hjärnstrukturer av särskild vikt vid födo- intagsreglering samt en central region för frisättning av noradrenalin hos mus. Coronin 7 påverkar också bananflugor på ett sådant sätt att de blir mot- ståndskraftiga mot svältperioder, utan att påverka deras generella aktivitet.

39 Acknowledgement

I would like to start by giving my special thanks to Prof. Helgi Schiöth for giving me the opportunity and privilege to work in his lab. I would also like to thank him for being my main supervisor and for the scientific help during my PhD. Robert Fredriksson for being my co-supervisor, your kindness and for providing great scientific advice in times of need. Special thanks to Michael Williams for great collaboration during my PhD. I am so thankful for all the effort and support you have provided. It has been a great pleasure working with you. Thank you for everything. I would also like to acknowledge Ashley Hutchinson, Vanni Caruso, Karin Nordenankar, and Pawel Olszewski for great scientific input. Thanks to all of the previous students who have helped me out with all the projects and experiments! Thanks to Madeleine Le Grevès for being an extraordinary organizer with the teaching. On the same topic, thanks to Emma Arvidsson, Hanna Pet- tersson and Calle Hallberg for amazing times in the basement during the animal course. It would not have been the same thing without you. I can honestly say that I miss those occasions. I would also like to thank Pleunie Hogenkamp, Maria Hägglund, Jessica Mwinyi, Galina Zheleznyakova and Claudia Pisanu for interesting scientific discussions and for being the best office mates ever. Emelie Perland, Sofie Hellsten and Sahar Roshanbin for being fun companions during my PhD. And of course everyone else in Helgi Lab who is not named in person! Special thanks to Lyle Wiemerslage for taking your time correcting my porli Inglish. Thanks to Birgitta for all the things you do around the lab. Martin Larhammar, häst, köttpirog and sauna. Kalicharan Patra, no words needed. Also, thanks to my previous supervisors during my master degree project Anders Enjin and Fatima Memic. To my coffee mate Niclas König, I miss our regular coffee at Bikupan. Oh, that reminds me - ½ thanks to Bikupan. Thanks to David Lagman for lots of gaming nights and “fun” spinning classes.

Thanks to all of my friends outside of work, David Rehnlund and Hotan Mojarradi. Thanks to Sascha Klasan, Sandra Castillo, Anders Kristof- fersson and Johan Nilsson (my homeboy) for poker nights and too many Caipirinhas. Karin Qvarnström for all the fika with interesting discussions and racist bikers.

40 Linda Solstrand Dahlberg, no words can truly express how thankful I am for everything that you have done for me and for all that we have experi- enced together. I am so glad I asked if you were working late that night in the stairway♥. Yes, the stairway. Also many thanks to my family: mamma, pappa, bror, Sara, Elis, Erik, Ascot, Rasmus, Charlie and Arve for giving me great support and pretend- ing to understand.

To all of you - Tack. Tack som fan.

41 References

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49 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1134 Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)

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